This application is a resubmission of a competitive renewal of HL102482 Variation in platelet function: the genetics of platelet gene expression. The work resulting from this funding included discoveries that (1) platelets from black subjects have greater PAR4-mediated platelet aggregation to thrombin, (2) a novel platelet protein, phosphatidylcholine transfer protein (PC-TP) contributed to this racial difference, and (3) the microRNA miR- 376c regulated PC-TP levels (Edelstein et al., Nature Medicine, 2013). Our pursuit of the molecular genetic basis of the racial difference in platelet aggregation led to the discovery of a common, racially divergent variant in F2RL3 (encodes PAR4) that induces an Ala120Thr substitution, accounting for ~50% of the racial difference in PAR4 reactivity. The PAR4 Thr120 variant is associated with greater PAR4-AP-induced platelet aggregation and IP generation in transfected cell lines. Additional preliminary data indicates that black race confers relative 3 resistance to COX and P2Y inhibition, and the Thr120 variant confers relative resistance to the FDA-approved 12 PAR1 inhibitor, vorapaxar, via enhanced thrombin responsiveness through PAR4. Furthermore, PAR4 Thr120- positive platelets - and especially homozygotes (40% of black subjects) - show resistance to inhibition by a novel PAR4 antagonist, YD-3. These data suggest that compared to white patients with cardiovascular disease, black patients may receive less benefit from most FDA-approved anti-platelet agents. The goals of this renewal application are to characterize the differential signaling, pharmacogenetic and clinical outcomes of F2RL3 variants, and identify novel PAR4 Thr120 antagonists. Aim 1 will characterize (a) differences in PAR4 Ala120Thr variant thrombin-induced signaling and shear-induced aggregation, and (b) the dominant-negative effects of a second PAR4 Phe296Val variant. Aim 2 will characterize the effect of PAR4 variants on thrombin- induced platelet aggregation in the presence of (a) anti-platelet agents (aspirin, P2Y inhibitor and vorapaxar) 12 and (b) a class of PAR4 inhibitors in clinical or pre-clinical development. Aim 3a will test for F2RL3 SNPs as risk factors for ischemic cardiovascular events in public GWASs of ~80,000 subjects. In Aim 3b we will genotype ~6,000 samples from the TRACER study and test for associations between F2RL3 SNPs and both ischemic cardiovascular and bleeding outcomes in patients receiving vorapaxar. Because current models of platelet signaling are likely based primarily on experiments using blood from white donors, and because we know the F2RL3 allele frequencies in all races/ethnic groups, successful completion of these Aims will substantively broaden basic understanding of platelet biology, develop variant-specific PAR4 inhibitors that could improve cardiovascular outcomes for PAR4 Thr120-positive patients (~86% African ancestry and 36% non-African ancestry), and may provide a rational basis for decisions about use and/or dosing of new PAR1 inhibitors based on patient race and/or F2RL3 genotypes.